The experiments described in this application are directed toward understanding the biochemical mechanisms by which SV40 large T transforms cells. Recently, three classes of large T mutants have been isolated which are central to this investigation: transformation defective (td) mutants; supertransforming (st) mutants which fail to bind with high affinity to wt SV40 origin sequences, and mutants which contain a defective nuclear location signal and therefore cannot localize large T to the nucleus (cytoplasmic mutants). The biochemical and biological activities of the large T encoded by these mutants will be investigated. Since data with cytoplasmic mutants has shown that large T need not be in the nucleus to transform established cell lines, transformed cells will be fractionated and the non-nuclear forms of large T examined. In particular, identification of the function affected in td mutants will be sought. The phosphorylation patterns of wt and mutant large Ts will be analyzed by protein fingerprinting to determine whether aberrant phosphorylation may serve as a marker of the td phenotype. All mutants will be tested for associated protein and phosphatidylinositol kinase activities. Any activity detected will be characterized to determine whether the mechanism of transformation by large T is connected, directly or indirectly, to the complex network of second messengers generated by the turnover of phosphatidylinositol. In order to find out whether the function affected by the td mutation is required for the immortalization as well as for transformation of cells in culture, or whether immortalizing and transforming functions are separable on the large T molecule, the viral DNA will be inserted into a retroviral vector containing an independently selectible marker and transfected into primary cells. The capacity of the cytoplasmic and st mutants to immortalize will also be tested in this way. It is hoped that the results of these investigations will provide some insight into the mechanism of transformation by SV40 large T.